Novel Staphylococcus aureus: Bacteriophage and Uses Thereof

ABSTRACT

The present invention is directed to isolated bacteriophage having strong lytic activity against strains of  Staphylococcus aureus,  in particular, MRSA, and methods of using that bacteriophage, and/or progeny or derivatives derived therefrom, to control the growth of  Staphylococcus aureus  in various settings.

FIELD OF THE INVENTION

The present invention relates to a novel bacteriophage designated SAML-4, SAML-150, and SATA 8505 (the “Deposited bacteriophage”), and compositions and preparations corresponding thereto. More specifically, isolated bacteriophage preparations possessing lytic activity against strains of Staphylococcus aureus (the “Targeted Bacteria”) are provided in order to control the growth of the Targeted Bacteria, which will reduce their ability to contaminate and colonize various environments, including but not limited to (i) raw, unprocessed food products, (ii) equipment used to process or manufacture various food products, (iii) various food products processed or manufactured with equipment contaminated with the Targeted Bacteria, (iv) animals contaminated with the Targeted Bacteria, and (v) animal environments contaminated with the Targeted Bacteria. The invention also provides methods for detecting the presence of the Targeted Bacteria in processed or unprocessed food products, and in equipment used to process or manufacture the food products. In addition, the invention provides methods of using the Deposited bacteriophage to remove the Targeted Bacteria from medical, veterinary, animal husbandry, and other environments where they may be passed to humans or animals. Also, the invention additionally provides methods of using the bacteriophage to prevent and treat animal and human diseases caused by the Targeted Bacteria.

BACKGROUND OF THE INVENTION

Bacteriophages are bacterial viruses that attach to their specific hosts and kill them by internal replication and bacterial lysis involving a complex lytic cycle involving several structural and regulatory genes. Phages are very specific in that they only attack their targeted bacterial hosts. They cannot infect human or other eukaryotic cells. Bacteriophages were first identified, in the early part of the 20th century by Frederick Twort and Felix D'Herelle who called them bacteriophages or bacteria-eaters (from the Greek phago meaning to eat or devour) (Duckworth, D. H. (1976). Who discovered bacteriophage? Bacteriol Rev 40(4): 793-802; Summers, W. C. (1999). Bacteriophage discovered. Felix d'Herelle and the origins of molecular biology. New Haven, Conn., Yale University Press: 47-59). At that time, with the age of antibiotics still in the future, bacteriophages were considered to be a potentially powerful cure for bacterial infections, and they were therapeutically utilized throughout the world during the pre-antibiotic era. The use of phages in humans was found to he very safe; however, phage therapy did not always work and, with the advent of antibiotics that were effective against a broad spectrum of pathogenic bacteria, it gradually fell out of favor in the United States and Western Europe. Several factors (reviewed in more detail in Sulakvelidze, A., Z., Alavidze, et al. (2001). Bacteriophage therapy. Antimicrob Agents Chemother 45(3): 649-659; Summers, W. C. (2001). Bacteriophage therapy. Ann Rev Microbiol 55: 437-51), including the lack of a broad understanding of phage biology and inadequate diagnostic bacteriology techniques, contributed to the failure of some of the early phage therapy studies and to the associated decline of interest in phage therapy in the West. At the same time, phage therapy continued to be utilized in the former Soviet Union and Eastern Europe, where phage therapy still is being used to treat a wide range of bacterial diseases ranging from intestinal infections to septicemia. Comprehensive information about human and veterinary applications of bacteriophages has been recently reviewed by several investigators (Alisky, J., K. Iczkowski, et al. (1998). Bacteriophages show promise as antimicrobial agents. J Infect 36(1): 5-15; Summers, W. C. (2001). Bacteriophage therapy. Annu Rev Microbiol 55: 437-51; Merril, C. R., D. Scholl, et al. (2003). “The prospect for bacteriophage therapy in Western medicine.” Nat. Rev Drug Discov 2(6): 489-497; Sulakvelidze, A. and P. Barrow (2005). Phage therapy in animals and agribusiness. Bacteriophages: Biology and Applications. E. Kutter and A. Sulakvelidze. Boca Raton, Fla., CRC Press: 335-380; Sulakvelidze, A. and E. Kutter (2005). Bacteriophage therapy in humans. Bacteriophages: Biology and Application. E. Kutter and A. Sulakvelidze, Boca Raton, Fla., CRC Press: 381-436).

Methicillin-resistant Staphylococcus aureus (MRSA) is a type of Staphylococcus aureus that is resistant to certain antibiotics, in particular methicillin. MRSA may also be resistant to other antibiotics including but not limited to oxacillin, penicillin, and amoxicillin. Some MRSA strains may also be resistant to vancomycin. Staphylococcal infections, including MRSA, occur most frequently among persons in hospitals and healthcare facilities (such as nursing homes and dialysis centers) who have weakened immune systems. MRSA infections that occur in otherwise healthy people who have not been recently (within the past year) hospitalized or had a medical procedure (such as dialysis, surgery, catheters) are known as community-associated (CA)-MRSA infections. MRSA is important because of its pathogenicity, limited treatment options, and MRSA are transmissible. “Healthcare-Associated Methicillin Resistant Staphylococcus aureus (HA-MRSA)” from the Centers for Disease Control website (2008).

MRSA is also used to describe multidrug-resistant S. aureus (MDRSA) strain, which is used to refer to a Staphylococcus aureus strain that is resistant to at least oxacillin, lincosamides, erythromycin, and chloramphenicol.

Despite the use of bacteriophage in various practical settings, including the treatment of diseases in various animals, there remains in the art a need for the discovery of novel bacteriophages, selection of optimal bacteriophages for specific practical applications, and identifying methods for using these bacteriophages in several critical areas, including clinical applications, food safety-related uses and environmental decontamination. For example, one significant need concerns the treatment of processed or unprocessed food products to reduce, eliminate or prevent colonization with undesirable bacteria such as pathogens responsible for food-borne illness and food spoilage organisms. A second critical area of need concerns the removal of undesirable bacteria from industrial environments such as food processing facilities to prevent colonization thereof. A third critical area of need concerns the removal of antibiotic resistant organisms from environments where they may be passed to susceptible humans and animals, such as hospitals, nursing homes, veterinary facilities, and other such environments. A fourth critical area of need is in the reduction or elimination of colonization of patients, healthcare workers, and other individuals by antibiotic-resistant pathogens, specifically including methicillin-resistant Staphylococcus aureus (MRSA) as well as MRSA strains that are also resistant to vancomycin. Additionally, new bacteriophage and methods of using the same are needed for the prevention or treatment of animal and human bacterial disease, particularly those diseases caused by antibiotic-resistant organisms.

SUMMARY OF THE INVENTION

The invention meets the described needs and more by providing compositions comprising novel SAML-4, SAML-150, and SATA 8505 bacteriophage having lytic specificity for the Targeted Bacteria. The invention additionally provides methods of using the Deposited bacteriophage to control or prevent the infection or colonization of processed and unprocessed food products by Targeted Bacteria, or colonization of equipment involved in the processing of the same food product(s). The invention additionally provides methods of using the Deposited bacteriophage to prevent, eradicate, or reduce the levels of colonization of various animals (including humans) with Targeted Bacteria. The invention also provides methods of detecting the presence of Targeted Bacteria cells on processed or unprocessed food products, or equipment involved in the processing of the same food products. The invention additionally provides methods of using the Deposited bacteriophage for the removal of antibiotic-resistant or other undesirable pathogens from medical, veterinary, animal husbandry, and other environments where they may be passed to humans or animals. The invention additionally provides for methods of using the Deposited bacteriophage to prevent or treat human and/or other animal diseases caused by Targeted Bacteria.

BRIEF DESCRIPTION OF THE FIGURES Figures

FIG. 1 shows a Restriction Fragment Length Polymorphism (RFLP) profile of the SAML-4, SAML-150, and SATA 8505 bacteriophage in comparison to four other bacteriophages also specific for the Targeted Bacteria. The Figure represents RFLP analysis of a EcoR I digest of genomic DNA from the indicated deposited bacteriophage. The lanes indicate: 1, DNA Ladder Mix (Fermentas; Lot 00020760, Cat #SM0331); 2-4, SATA-8505 (three different lots: 061808, 061708, and 061108, respectively); 5-7, SAML-150 (three different lots; 062508, 062008, and 061208, respectively); 8-10, SAML-4 (three different lots: 061908, 061608, and 061008, respectively). Gel: 1% Agarose.

Tables

Table 1 shows the lytic specificity of the bacteriophage for Staphylococcus aureus, the Targeted Bacteria.

TABLE 1 Lytic Specificity of the Deposited Bacteriophage Bacteria Strains Phage Strength: 2 × 10⁴ PFU/mL # Provider Intralytix ID SAML-4 SAML-150 SATA-8505 1 VA Center SA-1 + + + 2 VA Center SA-2 + + + 3 VA Center SA-3 + + − 4 VA Center SA-4 + − + 5 VA Center SA-5 + + + 6 Unknown SA-6 − − + 7 Unknown SA-7 + + + 8 Unknown SA-8 + − + 9 Unknown SA-9 − + − 10 Unknown SA-10 + + + 11 VA Center SA-11 − + + 12 VA Center SA-12 + + + 13 VA Center SA-13 + − − 14 VA Center SA-14 + + + 15 VA Center SA-15 + + + 16 VA Center SA-16 + + + 17 VA Center SA-17 + + + 18 VA Center SA-18 + − + 19 VA Center SA-19 + + + 20 VA Center SA-20 + − + 21 VA Center SA-21 + + + 22 VA Center SA-22 + + + 23 VA Center SA-23 + + + 24 VA Center SA-24 + − + 25 VA Center SA-25 + + + 26 VA Center SA-26 + + + 27 VA Center SA-27 − + − 28 VA Center SA-28 + + + 29 VA Center SA-29 + + + 30 VA Center SA-30 + + + 31 VA Center SA-31 + + + 32 VA Center SA-32 + + + 33 VA Center SA-33 + + + 34 VA Center SA-34 − − − 35 VA Center SA-35 + + + 36 ATCC SA-36 + + + 37 ATCC SA-37 + + + 38 VA Center SA-38 + + + 39 VA Center SA-39 + + + 40 VA Center SA-40 + + + 41 VA Center SA-41 + + + 42 VA Center SA-42 + + + 43 VA Center SA-43 + + + 44 VA Center SA-44 − − − 45 VA Center SA-45 + + + 46 VA Center SA-46 + + + 47 VA Center SA-47 + + + 48 VA Center SA-48 + + + 49 VA Center SA-49 + + + 50 VA Center SA-50 + + + 51 VA Center SA-51 + + + 52 VA Center SA-52 + + + 53 VA Center SA-53 + + + 54 VA Center SA-54 + + + 55 VA Center SA-55 + + + 56 VA Center SA-56 + + + 57 VA Center SA-57 + + + 58 VA Center SA-58 + + + 59 VA Center SA-59 + + + 60 VA Center SA-60 + − + 61 VA Center SA-61 + + + 62 VA Center SA-62 − − − 63 VA Center SA-63 + + + 64 VA Center SA-64 + + + 65 VA Center SA-65 + − − 66 VA Center SA-66 + + + 67 VA Center SA-67 + + + 68 VA Center SA-68 + + + 69 VA Center SA-69 + + + 70 VA Center SA-70 + + + 71 VA Center SA-71 − − − 72 VA Center SA-72 + + + 73 VA Center SA-73 + + + 74 VA Center SA-74 + + + 75 VA Center SA-75 + + + 76 VA Center SA-76 − + − 77 VA Center SA-77 − + − 78 VA Center SA-78 + + + 79 VA Center SA-79 + + + 80 VA Center SA-80 + + + 81 VA Center SA-81 + + + 82 VA Center SA-82 + + + 83 VA Center SA-83 + + + 84 VA Center SA-84 + + + 85 VA Center SA-85 + + + 86 VA Center SA-86 + − + 87 VA Center SA-87 − − − 88 VA Center SA-88 + − + 89 VA Center SA-89 + + + 90 VA Center SA-90 + + + 91 VA Center SA-91 missing strain 92 VA Center SA-92 + + + 93 VA Center SA-93 + + + 94 VA Center SA-94 + + + 95 VA Center SA-95 + + + 96 VA Center SA-96 − + − 97 VA Center SA-97 + + + 98 VA Center SA-98 + + + 99 VA Center SA-99 + + + 100 VA Center SA-100 + + + 101 VA Center SA-101 + + + 102 VA Center SA-102 + + + 103 VA Center SA-103 + + + 104 VA Center SA-104 + + + 105 VA Center SA-105 + + + 106 VA Center SA-106 + + + 107 VA Center SA-107 + + + 108 VA Center SA-108 + + + 109 VA Center SA-109 + + + 110 VA Center SA-110 missing strain 111 VA Center SA-111 + + + 112 VA Center SA-112 + + + 113 VA Center SA-113 + + + 114 VA Center SA-114 + + + 115 VA Center SA-115 + + + 116 VA Center SA-116 − − − 117 VA Center SA-117 − − − 118 VA Center SA-118 + + + 119 VA Center SA-119 + + + 120 VA Center SA-120 + − + 121 VA Center SA-121 + − + 122 VA Center SA-122 − − − 123 VA Center SA-123 + + + 124 VA Center SA-124 − + − 125 VA Center SA-125 − + − 126 VA Center SA-126 + + + 127 VA Center SA-127 + + + 128 VA Center SA-128 + + + 129 VA Center SA-129 − − − 130 VA Center SA-130 − + − 131 VA Center SA-131 − + − 132 VA Center SA-132 + + + 133 VA Center SA-133 − + − 134 VA Center SA-134 + − + 135 VA Center SA-135 + + + 136 VA Center SA-136 + + + 137 VA Center SA-137 + + + 138 VA Center SA-138 + + + 139 VA Center SA-139 + + + 140 VA Center SA-140 + − + 141 VA Center SA-141 + + + 142 VA Center SA-142 + + + 143 VA Center SA-143 + + + 144 VA Center SA-144 + + + 145 VA Center SA-145 + + + 146 VA Center SA-146 + − + 147 VA Center SA-147 + + + 148 VA Center SA-148 + + + 149 VA Center SA-149 + + + 150 VA Center SA-150 + + + 151 VA Center SA-151 − − + 152 VA Center SA-152 + + + 153 VA Center SA-153 + + + 154 VA Center SA-154 + + + 155 VA Center SA-155 + + + 156 VA Center SA-156 + + + 157 VA Center SA-157 + + + 158 VA Center SA-158 + − + 159 VA Center SA-159 − − − 160 VA Center SA-160 + − + 161 VA Center SA-161 + − + 162 VA Center SA-162 + + + 163 VA Center SA-163 − − − 164 VA Center SA-164 + + + 165 VA Center SA-165 + + + 166 VA Center SA-166 + + + 167 VA Center SA-167 + + + 168 VA Center SA-168 + + + 169 VA Center SA-169 + − − 170 VA Center SA-170 + − − 171 VA Center SA-171 − − − 172 VA Center SA-172 + + + 173 VA Center SA-173 + + + 174 VA Center SA-174 + + + 175 VA Center SA-175 + + + 176 VA Center SA-176 + + + 177 VA Center SA-177 + + + 178 VA Center SA-178 + + + 179 VA Center SA-179 + + + 180 VA Center SA-180 + + + 181 VA Center SA-181 + + + 182 VA Center SA-182 − − + 183 VA Center SA-183 + + + 184 VA Center SA-184 − + − 185 VA Center SA-185 − + − 186 VA Center SA-186 + + + 187 VA Center SA-187 − − − 188 VA Center SA-188 + + + 189 VA Center SA-189 + + + 190 VA Center SA-190 + + + 191 VA Center SA-191 − − − 192 VA Center SA-192 + + + 193 VA Center SA-193 + + + 194 VA Center SA-194 + + + 195 VA Center SA-195 + + + 196 VA Center SA-196 + + + 197 VA Center SA-197 + + + 198 VA Center SA-198 − − − 199 VA Center SA-199 − − − 200 VA Center SA-200 + + + 201 VA Center SA-201 + + + 202 VA Center SA-202 + + + 203 VA Center SA-203 + + + 204 VA Center SA-204 + + + 205 VA Center SA-205 + + + 206 VA Center SA-206 + + + 207 VA Center SA-207 + + + 208 VA Center SA-208 + + + 209 VA Center SA-209 + + + 210 Unknown SA-210 + + + 211 ATCC SA-211 + + + 212 Unknown SA-212 + + + 213 Unknown SA-213 + + + 214 Unknown SA-214 + + + 215 Unknown SA-215 − − − 216 Unknown SA-216 + + + 217 Unknown SA-217 + + + 218 Unknown SA-218 + + + 219 Unknown SA-219 + + + 220 Unknown SA-220 + + + 221 Unknown SA-221 + + + 222 Unknown SA-222 + − + 223 Unknown SA-223 + + + 224 Unknown SA-224 − − − 225 Unknown SA-225 + + + 226 Unknown SA-226 + + + 227 Unknown SA-227 + + + 228 Unknown SA-228 + + + 229 Unknown SA-229 − − − 230 Unknown SA-230 − − − 231 Unknown SA-231 − + − 232 Unknown SA-232 − + − 233 Unknown SA-233 + + + 234 Unknown SA-234 − − − 235 Unknown SA-235 + + + 236 Unknown SA-236 + − + 237 Unknown SA-237 − − − 238 Unknown SA-238 + − + 239 Unknown SA-239 + − + 240 Unknown SA-240 + + + 241 Unknown SA-241 − − − 242 Unknown SA-242 + + + 243 Unknown SA-243 + + + 244 Unknown SA-244 − − − 245 Unknown SA-245 + + + 246 Unknown SA-246 + + + 247 Unknown SA-247 + + + 248 Unknown SA-248 + + + 249 Unknown SA-249 + + + 250 Unknown SA-250 + − + 251 Unknown SA-251 + − + 252 Unknown SA-252 + + + 253 Unknown SA-253 + + + 254 Unknown SA-254 − − − 255 Unknown SA-255 + − + 256 Unknown SA-256 + + + 257 Unknown SA-257 − − − 258 Unknown SA-258 + + + 259 Unknown SA-259 + + + 260 Unknown SA-260 + + + 261 Unknown SA-261 + + + 262 Unknown SA-262 + + + 263 Unknown SA-263 + + + 264 Unknown SA-264 + − + 265 Unknown SA-265 + + + 266 Unknown SA-266 + + + 267 Texas--Dr. Randall Wolcott SA-267 − + − 268 Texas--Dr. Randall Wolcott S-268 − − − 269 Texas--Dr. Randall Wolcott S-269 − + + 270 Texas--Dr. Randall Wolcott S-270 − + − 271 Texas--Dr. Randall Wolcott SA-271 + + + 272 Texas--Dr. Randall Wolcott S-272 + − + 273 Texas--Dr. Randall Wolcott SA-273 + + + 274 Texas--Dr. Randall Wolcott SA-274 + + + 275 Texas--Dr. Randall Wolcott SA-275 − − − 276 Texas--Dr. Randall Wolcott SA-276 + − + 277 Texas--Dr. Randall Wolcott SA-277 + + + 278 Texas--Dr. Randall Wolcott S-278 − + − 279 Texas--Dr. Randall Wolcott SA-279 + + + 280 Texas--Dr. Randall Wolcott SA-280 − − + 281 Texas--Dr. Randall Wolcott SA-281 + + + 282 Texas--Dr. Randall Wolcott S?-282 + + + 283 Texas--Dr. Randall Wolcott SA-283 + + + 284 Texas--Dr. Randall Wolcott SA-284 + + + 285 Texas--Dr. Randall Wolcott SA-285 − − − 286 Texas--Dr. Randall Wolcott SA-286 + + + 287 Texas--Dr. Randall Wolcott SA-287 − + − 288 Texas--Dr. Randall Wolcott SA-288 + + + 289 Texas--Dr. Randall Wolcott S-289 − − − 290 Texas--Dr. Randall Wolcott SA-290 + + + 291 Texas--Dr. Randall Wolcott S-291 + − + 292 Texas--Dr. Randall Wolcott S-292 − − − 293 Texas--Dr. Randall Wolcott SA-293 + + + 294 Texas--Dr. Randall Wolcott S-294 + − + 295 Texas--Dr. Randall Wolcott SA-295 + + + 296 Texas--Dr. Randall Wolcott S-296 − − + 297 Merck research Lab. S-297 − − + 298 ATCC SA-298 + + + 299 ATCC SA-299 + + + 300 WCC-TEXAS Dan Rhoads SA-300 + + + 301 WCC-TEXAS Dan Rhoads SA-301 − − − 302 WCC-TEXAS Dan Rhoads SA-302 + + + 303 WCC-TEXAS Dan Rhoads SA-303 + + + 304 WCC-TEXAS Dan Rhoads S-304 − − − 305 WCC-TEXAS Dan Rhoads S-305 − − − 306 WCC-TEXAS Dan Rhoads SA-306 + − + 307 WCC-TEXAS Dan Rhoads S-307 + − + 308 WCC-TEXAS Dan Rhoads S-308 − − − 309 WCC-TEXAS Dan Rhoads SA-309 − − − 310 WCC-TEXAS Dan Rhoads SA-310 + + + 311 WCC-TEXAS Dan Rhoads SA-311 − + − 312 WCC-TEXAS Dan Rhoads S-312 − − − 313 WCC-TEXAS Dan Rhoads SA-313 − − − 314 WCC-TEXAS Dan Rhoads SA-314 + + + 315 WCC-TEXAS Dan Rhoads SA-315 + + + 316 WCC-TEXAS Dan Rhoads SA-316 + + + 317 WCC-TEXAS Dan Rhoads SA-317 + − + 318 WCC-TEXAS Dan Rhoads SA-318 + + + 319 WCC-TEXAS Dan Rhoads SA-319 + − + 320 WCC-TEXAS Dan Rhoads SA-320 + + + 321 WCC-TEXAS Dan Rhoads SA-321 + + + 322 WCC-TEXAS Dan Rhoads SA-322 − + − 323 WCC-TEXAS Dan Rhoads SA-323 + + + 324 WCC-TEXAS Dan Rhoads SA-324 + + + 325 WCC-TEXAS Dan Rhoads S-325 + + + 326 WCC-TEXAS Dan Rhoads SA-326 + + + 327 WCC-TEXAS Dan Rhoads SA-327 + + + 328 WCC-TEXAS Dan Rhoads SA-328 − − − 329 WCC-TEXAS Dan Rhoads SA-329 + + + 330 WCC-TEXAS Dan Rhoads S-330 − − − 331 WCC-TEXAS Dan Rhoads SA-331 − − − 332 WCC-TEXAS Dan Rhoads S-332 − − − 333 WCC-TEXAS Dan Rhoads SA-333 − − − 334 WCC-TEXAS Dan Rhoads SA-334 − − − 335 WCC-TEXAS Dan Rhoads SA-335 − − − 336 WCC-TEXAS Dan Rhoads SA-336 − − − 337 WCC-TEXAS Dan Rhoads SA-337 − − − 338 WCC-TEXAS Dan Rhoads SA-338 − − − 339 WCC-TEXAS Dan Rhoads SA-339 + + + 340 WCC-TEXAS Dan Rhoads SA-340 − − − 341 WCC-TEXAS Dan Rhoads SA-341 + + + 342 WCC-TEXAS Dan Rhoads SA-342 − − − 343 WCC-TEXAS Dan Rhoads SA-343 − − − 344 WCC-TEXAS Dan Rhoads SA-344 − − − 345 WCC-TEXAS Dan Rhoads SA345 − − − 346 WCC-TEXAS Dan Rhoads SA-346 + − + 347 WCC-TEXAS Dan Rhoads SA-347 + − + 348 WCC-TEXAS Dan Rhoads SA-348 + + + 349 WCC-TEXAS Dan Rhoads SA-349 + − + 350 WCC-TEXAS Dan Rhoads SA-350 + − + 351 WCC-TEXAS Dan Rhoads SA-351 − − − 352 WCC-TEXAS Dan Rhoads SA-352 − − − 353 WCC-TEXAS Dan Rhoads SA-353 + + + 354 WCC-TEXAS Dan Rhoads SA-354 + + + 355 WCC-TEXAS Dan Rhoads SA-355 + + + 356 WCC-TEXAS Dan Rhoads S-356 + − + 357 WCC-TEXAS Dan Rhoads SA-357 + + + 358 WCC-TEXAS Dan Rhoads SA-358 + − + 359 WCC-TEXAS Dan Rhoads SA-359 − − − 360 WCC-TEXAS Dan Rhoads SA-360 + + + 361 WCC-TEXAS Dan Rhoads SA-361 − + − 362 WCC-TEXAS Dan Rhoads SA-362 + − + 363 WCC-TEXAS Dan Rhoads SA-363 − − − 364 WCC-TEXAS Dan Rhoads SA-364 + − + 365 WCC-TEXAS Dan Rhoads SA-365 + − + 366 WCC-TEXAS Dan Rhoads SA-366 + − + 367 WCC-TEXAS Dan Rhoads SA-367 + − + 368 WCC-TEXAS Dan Rhoads SA-368 − − − 369 WCC-TEXAS Dan Rhoads SA-369 + + + 370 WCC-TEXAS Dan Rhoads SA-370 + + + 371 WCC-TEXAS Dan Rhoads S-371 − − − 372 WCC-TEXAS Dan Rhoads S-372 − − − 373 WCC-TEXAS Dan Rhoads S-373 − − − 374 WCC-TEXAS Dan Rhoads S-374 − − − 375 WCC-TEXAS Dan Rhoads S-375 − − − 376 WCC-TEXAS Dan Rhoads S-376 − − − 377 ATCC SA-377 + + + 378 ATCC SA-378 + − + 379 ATCC SA-379 − − − 380 ATCC SA-380 + + + 381 University of Naples Federico II SA-381 + − + 382 University of Naples Federico II SA-382 + − + 383 University of Naples Federico II SA-383 + + + 384 UTEP/UT SA-384 − − + 385 UTEP/UT SA-385 + + + 386 UTEP/UT SA-386 + + + 387 UTEP/UT SA-387 + + + 388 UTEP/UT SA-388 + + + 389 UTEP/UT SA-389 + + + 390 UTEP/UT SA-390 + + + 391 UTEP/UT SA-391 + − + 392 UTEP/UT SA-392 + + + 393 UTEP/UT SA-393 + + + 394 UTEP/UT SA-394 − + − 395 UTEP/UT SA-395 + + + 396 UTEP/UT SA-396 + + + 397 UTEP/UT SA-397 + + + 398 UTEP/UT SA-398 + + + 399 UTEP/UT SA-399 + − + 400 UTEP/UT SA-400 + + + 401 UTEP/UT SA-401 + + + 402 UTEP/UT SA-402 + + + 403 UTEP/UT SA-403 + + + 404 UTEP/UT SA-404 + + + 405 UTEP/UT SA-405 + + + 406 UTEP/UT SA-406 + + + 407 UTEP/UT SA-407 + − + 408 UTEP/UT SA-408 − − + 409 UTEP/UT SA-409 + − + 410 UTEP/UT SA-410 + − + 411 UTEP/UT SA-411 + − + 412 UTEP/UT SA-412 + − + 413 UTEP/UT SA-413 + − + 414 UTEP/UT SA-414 + − + 415 UTEP/UT SA-415 + − + 416 UTEP/UT SA-416 + − + 417 UTEP/UT SA-417 + − + 418 UTEP/UT SA-418 + − + 419 UTEP/UT SA-419 + + + 420 UTEP/UT SA-420 − − − 421 UTEP/UT SA-421 + − + 422 UTEP/UT SA-422 + − + 423 UTEP/UT SA-423 + + + 424 UTEP/UT SA-424 + − + 425 UTEP/UT SA-425 + − + 426 UTEP/UT SA-426 + − + 427 UTEP/UT SA-427 + − + 428 UTEP/UT SA-428 + − + 429 UTEP/UT SA-429 − + − 430 UTEP/UT SA-430 + + + 431 UTEP/UT SA-431 − + + 432 UTEP/UT SA-432 + + + 433 UTEP/UT SA-433 − + + 434 UTEP/UT SA-434 + + + 435 UTEP/UT SA-435 − + − 436 UTEP/UT SA-436 + − + 437 UTEP/UT SA-437 + − + 438 UTEP/UT SA-438 + − + 439 UTEP/UT SA-439 + − + 440 UTEP/UT SA-440 + + + 441 UTEP/UT SA-441 + + + 442 UTEP/UT SA-442 + + + 443 UTEP/UT SA-443 + + + 444 UTEP/UT SA-444 + − + 445 UTEP/UT SA-445 + + + 446 UTEP/UT SA-446 + − + 447 UTEP/UT SA-447 + − + 448 UTEP/UT SA-448 + − + 449 UTEP/UT SA-449 − + + 450 UTEP/UT SA-450 + − + 451 UTEP/UT SA-451 + − + 452 UTEP/UT SA-452 + + + 453 UTEP/UT SA-453 + + + 454 UTEP/UT SA-454 + + + 455 UTEP/UT SA-455 + + + 456 UTEP/UT SA-456 + + + 457 UTEP/UT SA-457 + + + 458 UTEP/UT SA-458 + + + 459 UTEP/UT SA-459 − + − 460 UTEP/UT SA-460 + − + 461 UTEP/UT SA-461 + − + 462 UTEP/UT SA-462 + − + 463 UTEP/UT SA-463 + + + 464 UTEP/UT SA-464 − + − 465 UTEP/UT SA-465 + + + 466 UTEP/UT SA-466 + − + 467 UTEP/UT SA-467 + − + 468 UTEP/UT SA-468 + − + 469 UTEP/UT SA-469 + + + 470 UTEP/UT SA-470 + − + 471 UTEP/UT SA-471 + + + 472 UTEP/UT SA-472 + − + 473 UTEP/UT SA-473 + + + 474 UTEP/UT SA-474 − − − 475 UTEP/UT SA-475 + − + 476 UTEP/UT SA-476 + − + 477 UTEP/UT SA-477 + − + 478 UTEP/UT SA-478 + + + 479 UTEP/UT SA-479 − − − 480 UTEP/UT SA-480 − − − 481 UTEP/UT SA-481 − − − 482 UTEP/UT SA-482 − + − 483 Eurofins Medinet SA-483 − − − 484 Eurofins Medinet SA-484 + − + 485 Eurofins Medinet SA-485 + − + 486 Eurofins Medinet SA-486 + − + 487 Eurofins Medinet SA-487 − − − 488 Eurofins Medinet SA-488 + + + 489 Eurofins Medinet SA-489 + + + 490 Eurofins Medinet SA-490 + − + 491 Eurofins Medinet SA-491 + − + 492 Eurofins Medinet SA-492 − − − 493 Eurofins Medinet SA-493 + + + 494 Eurofins Medinet SA-494 + + + 495 Eurofins Medinet SA-495 + − + 496 Eurofins Medinet SA-496 − − − 497 Eurofins Medinet SA-497 + − + 498 Eurofins Medinet SA-498 + − + 499 Eurofins Medinet SA-499 − − − 500 Eurofins Medinet SA-500 + + + 501 Eurofins Medinet SA-501 + + + 502 Eurofins Medinet SA-502 + + + 503 Eurofins Medinet SA-503 + − + 504 Eurofins Medinet SA-504 + + + 505 Eurofins Medinet SA-505 + + + 506 Eurofins Medinet SA-506 + − + 507 Eurofins Medinet SA-507 + − + 508 Eurofins Medinet SA-508 − − − 509 Eurofins Medinet SA-509 + + + 510 Eurofins Medinet SA-510 + + + 511 Eurofins Medinet SA-511 − − − 512 Eurofins Medinet SA-512 + + + 513 Eurofins Medinet SA-513 + + + 514 Eurofins Medinet SA-514 − − − 515 Eurofins Medinet SA-515 − − + 516 Eurofins Medinet SA-516 + + + 517 Eurofins Medinet SA-517 + − + 518 Eurofins Medinet SA-518 + − + 519 Eurofins Medinet SA-519 + − + 520 Eurofins Medinet SA-520 + + + 521 Eurofins Medinet SA-521 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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

As used herein, “isolated” will mean material removed from its original environment in which it naturally occurs, and thus is altered by the hand of man from its natural environment. Isolated material may be, for example, foreign nucleic acid included in a vector system, foreign nucleic acid contained within a host cell, or any material which has been removed from its original environment and thus altered by the hand of man. Isolated material further encompasses bacteriophage specific for the Targeted Bacteria or particular Targeted Bacteria isolates, isolated and cultured separately from the environment in which it was located, where these isolates are present in purified compositions that do not contain any significant, amount of other bacteriophage or bacterial strains, respectively.

As used therein, “Deposited bacteriophage” will mean isolated bacteriophage SAML-4, SAML-150, and SATA 8505 deposited with the ATCC on Sep. 8, 2008, and receiving ATCC Deposit Accession Nos. ______, ______, and ______.

As used therein, “Targeted Bacteria” will mean Staphylococcus aureus. In one embodiment, the Target Bacteria encompasses methicillin-resistant Staphylococcus aureus (MRSA) strain (i.e., Staphylococcus aureus that is resistant to certain antibiotics, in particular methicillin. MRSA may also be resistant to other antibiotics including but not limited to oxacillin, penicillin, and amoxicillin.) In another embodiment, the Target Bacteria encompasses multidrug-resistant S. aureus (MDRSA) strain (i.e., Staphylococcus aureus that is resistant to at least oxacillin, lincosamides, erythromycin, and chloramphenicol.) In another embodiment, the Staphylococcus aureus strain is resistant to vancomycin.

As used herein, “progeny” shall mean all bacteriophages, including descendants of Deposited bacteriophage or by other means well known in the art, whose RFLP profiles are substantially equivalent, to the RFLP profile of the Deposited bacteriophage in accordance with the standards advanced by Tenover et al. from the Linked States Centers for Disease Control and Prevention [Tenover, F. C. et al. (1995) Interpreting Chromosomal DNA Restriction Patterns Produced by Pulsed-Field Gel Electrophoresis: Criteria for Bacterial Strain Typing. J. Clin. Microbiol. 33:2233-2239, herein incorporated by reference]. Tenover et al. teaches the acceptable levels of variation that may be seen when the genomes identical propagated bacteriophages are electrophoretically analyzed following restriction enzyme digestion. Further, the term “progeny” encompasses bacteriophages which have accumulated minor genetic variations due to genetic drift (e.g., single nucleotide polymorphisms, conservative mutations, single base pair changes, silent mutations, deletion of DNA) due to, for instance, subsequent, serial, and/or successive passaging.

As used herein, “recombinant phage” shall mean ail modified versions of the Deposited bacteriophage or their progeny, obtained by serial passaging (in vivo or in vitro) or genetic manipulations of Deposited bacteriophage or their progeny. Such manipulations include, but are not limited to, introducing genes or gene cassettes encoding alternative proteins or nonfunctional proteins, or noncoding nucleotide sequences into the genome of either the Deposited bacteriophage.

As used herein, “derivatives” shall mean all substances that constitute subunits or expression products of the Deposited bacteriophage or their progeny, including (but not limited to) phage nucleic acids, partial or complete phage genes, gene expression products, and structural components (e.g., head, collar, sheath, base plate, phage coat, and tail fibers). For example, derivatives of the invention mean polyribonucleotide(s) and polydeoxyribonucleotide(s), including modified or unmodified bacteriophage DNA, cDNA, mRNA and synthetic polynucleotide sequences, as well as DMA/RNA hybrids. Polynucleotides of the invention also encompass modified polynucleotides, such as for example phosphorylated DNAs.

As used herein, “substantially pure” will mean material essentially free of any similar macromolecules or other biological entities that would normally be found with it in nature. For example, a substantially pure bacteriophage is in a composition that contains no more than 1% of other bacteriophages.

As used herein, “substantially equivalent” refers broadly to “indistinguishable” (i.e., bacteriophages that are genetically indistinguishable if their restriction patterns have the same numbers of bands and the corresponding bands are the same apparent size) and/or “closely related” (i.e., bacteriophages which where the PFGE pattern differs from the outbreak pattern by changes consistent with a single genetic event, i.e., a point mutation or an insertion or deletion of DNA) as set forth in Tenover, et at. (“Tenover”) which describes a system for interpreting chromosomal DNA Restriction Enzyme digest patterns (“RFLP”) using Pulsed-Field Gel Electrophoresis (PFGE). In particular, “substantially equivalent” encompasses bacteriophages which are indistinguishable front or closely related to each other that share the saute target specificity.

As used herein, “bacteriophage composition” will mean a composition comprising, or alternatively consisting essentially of, or alternatively consisting of, the Deposited bacteriophage. A “bacteriophage composition” as used herein does not include the Deposited bacteriophage as it exists in its natural environment prior to isolation and/or substantial purification.

As used herein, “colonization” or “colonized” will refer to the presence of Targeted Bacteria on foodstuff(s), or environmental surface(s), or in vivo such as in the gastrointestinal tract or skin of a mammalian organism without perceptible significant alteration other than the presence of bacteria. The terms “colonization” and “colonized” stand in contrast to the terms “infection” or “infected” which are commonly understood to require perceptible deleterious alteration as past of their definition, “Colonization” and “colonized” may also refer to the presence of bacteria in or on a human or animal without perceptible damage, alteration, or disease.

As used herein, “ATCC” will mean the American Type Culture Collection, located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA.

As used herein, “ORF” will mean an Open Reading Frame which is an in-frame sequence of codons that (in view of the genetic code) correspond to or encode a protein or peptide sequence. Two ORFs correspond to each other if the sequences or their complementary sequences encode the same amino acid sequences. An ORF sequence, operably associated with appropriate regulatory sequences, may be transcribed and translated into a polypeptide in vivo. A polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.

The Deposited Bacteriophage

The Deposited Bacteriophage has binding specificity for Targeted Bacteria, and is capable of lysing Targeted Bacteria. The invention further contemplates variants of the Deposited Bacteriophage, which are bacteriophage having minor variation(s) in the genomic sequence and polypeptides encoded thereby while retaining the same general genotypic and phenotypic characteristics as the Deposited Bacteriophage. Such variants are considered to be the Deposited Bacteriophage in accordance with the standards advanced by Tenover et al. from the United States Centers for Disease Control and Prevention (Tenover, F. C. et al. (1995) Interpreting Chromosomal DNA Restriction Patterns Produced by Pulsed-Field Gel Electrophoresis: Criteria for Bacterial Strain Typing. J. Clin. Microbiol. 33:2233-2239). The invention also contemplates progeny and bacteriophage derivative(s).

The invention contemplates the use of the Deposited Bacteriophage, and its progeny and derivatives, to control the growth on, or colonization of, processed and unprocessed food products by Targeted Bacteria, or the colonization of buildings and equipment, particularly those associated with the processing of the same food product. The invention also provides methods of identifying Targeted Bacteria as a bacterial diagnostic and/or detecting the presence of Targeted Bacteria on processed or unprocessed food products, or equipment or buildings such as those involved in the processing of the same food products. The invention further provides methods of using the Deposited Bacteriophage for the removal of antibiotic-resistant or other undesirable pathogens from medical, veterinary, animal husbandry, or any additional environments where they may be passed to humans or animals. The invention additionally provides for methods of using the Deposited Bacteriophage to prevent and/or treat human and animal diseases caused by Targeted Bacteria. The Deposited Bacteriophage is administered for the methods of the invention as a homogenous phage administration, or alternatively as a component of a multi-phage composition comprising several bacteriophages. These methods of use are provided with greater particularity infra.

Use of the Deposited Bacteriophage and its Progeny Food Preservation

In one embodiment, the invention contemplates a method for the prevention of foodborne illnesses caused by the Targeted Bacteria, comprising contacting a food product or products with a microbial growth inhibiting effective amount of a bacteriophage composition comprising the Deposited Bacteriophage. The modes of contact include, but are not limited to, spraying or misting the Deposited Bacteriophage composition on the food product(s), or by dipping or soaking the food product(s) in a solution containing a concentration of the Deposited Bacteriophage sufficiently high to inhibit the growth of Targeted Bacteria, or adding, injecting or inserting the Deposited Bacteriophage into the food product(s).

In another embodiment, the invention contemplates the application of the Deposited Bacteriophage composition to equipment associated with the processing of food produces), such as cutting instruments, conveyor belts, and any other implements utilized in the mass production of food products, including the preparation, storage and packaging steps of food processing. The Deposited Bacteriophage can additionally be introduced into packaging materials used to contain food product(s), prior to or following transfer of the food product(s) to the packaging materials. Alternatively the Deposited Bacteriophage can be useful in the local processing of food products located, for example, in the home or in a restaurant kitchen, using the same modes of contact as described supra.

In another embodiment of the invention, the Deposited Bacteriophage is added as a component of paper products, either during processing or after completion of processing of the paper products. Paper products to which the Deposited Bacteriophage may be added include, but are not limited to, paper towels, toilet paper, moist paper wipes. In a preferred embodiment of the invention, the Deposited Bacteriophage is added as a component of cleansing wipes. The Deposited Bacteriophage may be added in an aqueous state to a liquid-saturated paper product, or alternatively may be added in powder form, such as lyophilized, to dry paper products, or any combination thereof. In similar manner, the Deposited Bacteriophage may be incorporated into films such as those used for packaging foods, such as by impregnating or coating the film.

The methods of the invention further contemplate the application of the Deposited Bacteriophage to the floors, walls, ceilings, drains, or other environmental surfaces in structures such as the industrial food processing, military, or home environments. In a particularly preferred embodiment of the invention, the Deposited Bacteriophage is applied to refrigerated devices used to store or transport food or food products, including but not limited to, home and industrial refrigerators, deli-meat and cheese counters, refrigerated trucks, and mobile food-service vehicles.

In a non-limiting embodiment of the invention, the Deposited Bacteriophage of the invention is useful in preventing the colonization of, or inhibiting the growth of, Targeted Bacteria on processed or unprocessed food products by infecting, lysing or inactivating Targeted Bacteria present on said food product. Processed or unprocessed food products in which the Deposited Bacteriophage is particularly useful in preventing the growth or colonization of Targeted Bacteria include, but are not limited to beef (particularly ground beef), food products made with ground beef such as hamburgers, sloppy joes, lasagna, stews, and other ground beef preparations, fresh vegetables exposed to Targeted Bacteria presumably via animal waste, such as lettuce, spinach, green onions, and other fresh vegetables commonly grown out of doors in fields, drinking water, and foodstuffs secondarily contaminated with Targeted Bacteria through contact with contaminated foods, sewage, or animal feces.

The Deposited Bacteriophage can also be administered to potable and non-potable water sources to reduce or eliminate the presence of Targeted Bacteria.

Bacteriophage compositions of the invention may be provided in aqueous or non-aqueous embodiments for the preservation of food.

Aqueous embodiments of the Deposited Bacteriophage include aqueous compositions comprising, or alternatively consisting of, the Deposited Bacteriophage alone or in combination with other bacteriophage or bacteriophages. Aqueous embodiments of the Deposited Bacteriophage are available in solutions that include, but are not limited to, phosphate buffered saline, Luria-Bertani Broth or chlorine-free water.

Non-aqueous embodiments of the Deposited Bacteriophage include, but are not limited to, lyophilized compositions or spray-dried compositions comprising, or alternatively consisting of, the Deposited Bacteriophage alone or in combination with other bacteriophage(s). Freeze-dried and spray-dried compositions may also include soluble and/or insoluble carrier materials as, for example, processing aids.

The Deposited Bacteriophage can be administered at a concentration effective to prevent the initial colonization of foods with Targeted Bacteria, or to inhibit the growth or colonization of food or food products, as well as the equipment used to process or store food. In a non-limiting embodiment of the invention, the Deposited Bacteriophages typically administered at a growth inhibiting effective amount of a concentration of about 10⁷ to about 10¹¹ Plaque Forming Units (PFU)/ml. One of skill in the art is capable of ascertaining bacteriophage concentrations using widely known bacteriophage assay techniques (Adams, M. H. (1959). Methods of study bacterial viruses. Bacteriophages. London, Interscience Publishers, Ltd.: 443-519.). The Deposited bacteriophage at such concentrations may be applied at, for example, about 1 ml/500 cm² of food surface.

Environmental Control

In another embodiment of the invention, the Deposited Bacteriophage is administered to environments to control the growth or viability of Targeted Bacteria. Environments in which the Deposited Bacteriophage is useful to control the growth or viability of Targeted Bacteria include, but are not limited to, abattoirs, meat processing facilities, feedlots, vegetable processing facilities, medical facilities (including hospitals, out-patient clinics, school and/or university infirmaries, and doctors offices), military facilities, veterinary offices, animal husbandry facilities, public and private restrooms, and nursing and nursing home facilities. The invention further contemplates the use of the Deposited Bacteriophage for the battlefield decontamination of food stuffs, the environment, and personnel and equipment, both military and non-military,

The Deposited Bacteriophage is additionally useful alone or in combination with other bacteriophage(s) and/or other compounds, for preventing the formation of biofilms, or controlling the growth of biofilms, in various environments. Aqueous embodiments of the Deposited Bacteriophage are available in solutions that include, but are not limited to, phosphate buffered saline, Luria-Bertani Broth or chlorine-free water. In a particularly preferred embodiment, the Deposited Bacteriophage is used to control biofilm formation and growth in municipal water systems, industrial water systems, and personal water systems, as well as biofilms present in refrigerated environments.

The modes of administration include, but are not limited to, spraying, hosing, and any other reasonable means of dispersing aqueous or non-aqueous Bacteriophage compositions, in an amount sufficiently high to inhibit the growth or viability of Targeted Bacteria. In a non-limiting embodiment of the invention, the Deposited Bacteriophage is useful in preventing the growth or viability of Targeted Bacteria by infecting, lysing or inactivating Targeted Bacteria present in said environment. Administration of the Deposited Bacteriophage composition includes application to the floors, walls, counter-tops, ceilings, drains or any other environmental surface.

Bacteriophage compositions of the invention are available in aqueous or non-aqueous embodiments discussed earlier for Food Preservation applications.

In another embodiment of the invention, the Deposited Bacteriophage is added as a component of paper products, either during processing or after completion of processing of the paper products. Paper products to which the Deposited Bacteriophage may be added include, but are not limited to, paper towels, toilet paper, and moist paper wipes. In a preferred embodiment of the invention, the Deposited Bacteriophage is added as a component of cleansing wipes; it may be added in an aqueous state to a liquid-saturated paper product, or alternatively may be added in powder form such as a lyophilized preparation, to dry paper products, or any combination thereof.

The Deposited Bacteriophage can be administered at a concentration effective to inhibit the growth or viability of Targeted Bacteria in a particular environment. In a non-limiting embodiment of the invention, the Deposited Bacteriophage is administered at a concentration of about 10⁷ to 10¹¹ PFU/ml. One of skill in the art is capable of ascertaining bacteriophage concentrations using widely known bacteriophage assay techniques (Adams, M. H. (1959). Methods of study bacterial viruses. Bacteriophages. London, Interscience Publishers, Ltd.: 443-519.).

Prevention or Treatment of Infection or Colonization

In another embodiment, the invention contemplates a method for the prevention or treatment of illnesses caused by the Targeted Bacteria, comprising contacting a microbial growth inhibiting effective amount of a bacteriophage composition comprising the Deposited Bacteriophage with a site or sites of infection of a host mammal infected with Targeted Bacteria.

The infected mammalian host may be a human host or animal host. In particular, the host may be a bovine, poultry, or porcine host. Prevention of the infection by Targeted Bacteria, or treatment of infected persons or animals, is particularly preferred in immuno-compromised persons, pregnant females, and newborns and infants, who maybe at an elevated risk of Infection by Targeted Bacteria. The modes of contact include, but are not limited to, spraying or misting the bacteriophage composition on the infected mammalian host, by injecting at a site or sites of infection a pharmaceutically acceptable composition containing a concentration of the Deposited Bacteriophage sufficiently high to inhibit the growth of Targeted Bacteria, or by ingesting a solution containing a concentration of the Deposited Bacteriophage sufficiently high to inhibit the growth of Targeted Bacteria. Additional routes of administration include but are not limited to oral, rectal, topical, ophthalmic, buccal, intravenous, otic, nasal, vaginal, inhalation, and intrapleural.

In another nonlimiting embodiment of the invention, the Deposited Bacteriophage is useful for preparing bacterial vaccines or bacterins that eliminate or reduce colonization of the Targeted Bacteria in, and/or their being shed by, various agriculturally-important animals. One example of a practical application for that type of vaccine is in the cattle-raising industry, where its administration may significantly reduce colonization of cattle with the Targeted Bacteria; thus, improving public safety by reducing contamination of beef with the Targeted Bacteria.

Bacteriophage compositions of the invention are available in aqueous or non-aqueous embodiments discussed earlier for Food Preservation applications.

The Deposited Bacteriophage can be administered at a concentration effective to inhibit the growth or viability of Targeted Bacteria in the infected host. In a non-limiting embodiment of the invention, the Deposited Bacteriophage is administered at a concentration of about 10⁷ to 10¹¹ PFU/ml. One of skill in the art is capable of ascertaining bacteriophage concentrations using widely known bacteriophage assay techniques (Adams, M. H. (1959). Methods of study bacterial viruses. Bacteriophages. London, Interscience Publishers, Ltd.: 443-519.)

Depending on the severity of peculiarities of the infection, the Deposited Bacteriophage can be administered to animals (including humans) (i) orally, in tablet or liquid formulation (10⁵-10¹¹ PFU/dose), (ii) rectally, (iii) locally (skin, eye, ear, nasal mucosa, etc.), in tampons, rinses and creams, (iv) as aerosols or intrapleunal injections and (v) intravenously.

Use of Recombinant Bacteriophage

In one embodiment of the invention, homologous recombination techniques are used to introduce sequences encoding alternative proteins, non-functional proteins, or non-coding sequences into the bacteriophage DNA sequence. Such techniques are useful to “knock-out” undesired traits of the Deposited Bacteriophage, or alternatively to introduce different traits. In a particularly preferred embodiment of the invention, homologous recombination is used to “knock-out” ORFs encoding proteins that maybe involved in a lysogenic cycle of the bacteriophage.

In another embodiment of the invention, homologous recombination is used to introduce or knock-out genes involved in burst size. For example, homologous recombination is used to introduce alternative bacteriophage genes which delay the burst event or increase the phage burst size. References disclosing alternative bacteriophage genes involved in the timing of the burst event or the size of the phage burst include, but are not limited to (Johnson-Boaz, R., C. Y. Chang, et al. (1994). “A dominant mutation in the bacteriophage lambda S gene causes premature lysis and an absolute defective plating phenotype.” Mol Microbiol 13(3): 495-504; Wang, I. N., D. L. Smith, et al. (2000). “Holins: the protein clocks of bacteriophage infections.” Annu Rev Microbiol 54: 799-825).

In another embodiment of the invention, recombinant bacteriophage harboring reporter system(s) is generated for various practical applications. One example of possible application of such system is species identification/confirmation of Targeted Bacteria as bacterial diagnostics. Another possible application is the detection of the presence of viable cells of Targeted Bacteria to which the Deposited bacteriophage have specificity. Following the techniques of Loessner et al., for example, one of skill in the art can generate recombinant reporter bacteriophage (Loessner, M. J., C. E. Rees, et al. (1996), “Construction of luciferase reporter bacteriophage A511::luxAB for rapid and sensitive detection of viable Listeria cells” Appl Environ Microbiol 62(4): 1133-1140.). For example, the Vibrio harveyi luxAB gene may be introduced into the bacteriophage DNA sequence using techniques such as homologous recombination. An ideal target for the introduction of the luxAB gene is immediately downstream and in frame with an ORF encoding bacteriophage capsid protein, thereby creating a sequence encoding a fusion protein. The preferable location of introduction of the luxAB gene sequence is particularly before any sequence encoding a transcriptional terminator downstream of the ORF encoding a capsid protein. Other bacteriophage ORF sequences which may function as useful sources of luxAB gene-fusions include gene sequences encoding tail-sheath proteins, or any other late gene region sequences encoding phage head or tail proteins. The resulting recombinant bacteriophage may be used with methods of the invention to detect the presence of viable cells of Targeted Bacteria.

In addition to the Vibrio harveyi luxAB gene, other reporter genes which are useful for the generation of reporter bacteriophage include, but are not limited to, the firefly luciferase gene.

The invention further contemplates the introduction of one or more of the above-described recombinant events. For example, a recombinant bacteriophage of the invention may harbor one or more reporter gene(s) as well as lack one or more genes associated with certain undesirable biological functions of the bacteriophage.

Use of Bacteriophage Derivatives

Derivatives, such as polypeptides, including but not limited to bacteriophage lytic enzymes, encoded by the bacteriophage or die bacteriophage progeny are used for applications designed to prevent the growth of Targeted Bacteria through cell wall lysis. In this context, lytic polypeptides are useful for the prevention of the growth of Targeted Bacteria on processed and unprocessed food products, as well as equipment used for the processing of said food products.

In another preferred embodiment of the invention, bacteriophage derivatives are useful for the treatment of one or more infections in a mammal, including humans, by administering their therapeutically effective amounts to the patient. This method is useful for the treatment of infections of the gastrointestinal system. Similarly, this method is useful in a prophylactic setting for the prevention of infection, by Targeted Bacteria in pregnant mammals, including humans. This method of treatment is further useful for the prevention or other disorders or infections caused by Targeted Bacteria, such as acute bloody or non-bloody diarrhea, sometimes associated with hemolytic-uremic syndrome.

Another nonlimiting embodiment of the invention is that, the bacteriophage derivatives such as lysins will be useful for preparing bacterial vaccines or bacterins that eliminate or reduce colonization of the Targeted Bacteria in, and/or their being shed by, various agriculturally-important animals. One example of a practical application for that type of vaccine is in the cattle-raising industry, where administration of such vaccines/bacterins may significantly reduce colonization of cattle with the Targeted Bacteria; thus, improving public safety by reducing contamination of beef with the Targeted Bacteria.

Detection Systems

The Deposited bacteriophage, its progeny, recombinant bacteriophage, or derivatives of the above are useful in methods of screening environmental samples (including food products and food processing equipment) and clinical specimens for the presence of viable cells of Targeted Bacteria. For example, in one such system, recombinant bacteriophage containing a reporter system such as, for example, a luciferase reporter system is applied to the sample and analyzed at some time point in the future for the activation of the reporter molecule. The activation of the reporter molecule is indicative of the presence of viable cells of Targeted Bacteria.

The Deposited bacteriophage, its progeny, recombinant bacteriophage, or derivatives such as lytic enzymes are useful in methods of screening environmental samples including food products and food processing equipment and clinical specimens for the presence of viable cells of Targeted Bacteria, by monitoring and measuring bacterial metabolism products such as bacterial adenosine kinase (AK) or adenosine triphosphate (ATP) released as a result of specific lysis of Targeted Bacteria. For example, when the released ATP is incubated with a luciferin/luciferase mixture, a rapid flash of peak light emission occurs within less than a second, followed by a steady glow lasting for several hours. By measuring the luminescence, it is possible to obtain a quantitative estimate of the number of bacterial cells in a sample. Although the basic approach involved in such detection-based assays is fairly well-established, the existing assays have shortcomings that hinder their wide acceptance. For example, the various reagents that have been used to lyse bacteria and release their ATP have broad-specificity; therefore, ATP is released from all susceptible bacterial and eukaryotic cells present in the sample, which can cause false-positive readings. In this context, the original Deposited Bacteriophage, its progeny, recombinant bacteriophage, or derivatives such as lytic enzymes will specifically lyse Targeted Bacteria without affecting any other prokaryotic or eukaryotic cells that may be present in the sample, thus providing means for accurately arid specifically identifying and detecting Targeted Bacteria.

Epidemiological Typing

The Deposited Bacteriophage, and/or its progeny and derivatives may be further useful as a tool for the epidemiological typing of Targeted Bacteria. For example, one of skill in the art can use the Deposited Bacteriophage of the invention to screen a panel of Targeted Bacteria isolates to aid in the taxonomic identification of the Targeted Bacteria, by determining which isolates yield a positive lytic reaction to the Deposited Bacteriophage. For example, see (van der Mee-Marquet, N., M. Loessner, et al, (1997). “Evaluation of seven experimental phages for inclusion in the international phage set for the epidemiological typing of Listeria monocytogenes.” Appl Environ Microbiol 63(9): 3374-3377.).

Preparation of Vaccines or Bacterins

The Deposited Bacteriophage, and/or its progeny and derivatives, also may be valuable for preparing bacterial lysates to be used as vaccines or bacterins. The immunogenicity of such vaccines or bacterins may be superior to that of so-called dead cell vaccines because phage-mediated lysis is a more effective and gentler approach for exposing protective antigens of bacteria than are approaches used to prepare the latter vaccines. For example, methods commonly used to inactivate bacterial pathogens for dead-cell vaccines, including but not limited to heat treatment, UV-irradiation, and chemical treatment, may deleteriously affect a vaccine's effectiveness by reducing the antigenicity of relevant immunological epitopes (Holt, M. E., M. R. Enright, et al. (1990). “Immunisation of pigs with killed cultures of Streptococcus suis type 2.” Res Vet Sci 48(1): 23-27., Melamed, D., G. Leitner, et al. (1991). “A vaccine against avian colibacillosis based on ultrasonic inactivation of Escherichia coli.” Avian Dis 35(1): 17-22.; Lauvau, G., S. Vijh, et al. (2001). “Pruning of memory but not effector CD8 T cells by a killed bacterial vaccine.” Science 294(5547): 1735-1739). The presence of viable bacteriophage may also serve as an additional efficacy-enhancing factor, increasing the effectiveness of a phage lysate via their antibacterial effect on the Targeted Bacteria.

EXAMPLES Example 1 SAML-4, SAML-150, and SATA 8505 Bacteriophage Isolation

The SAML-4, SAML-150, and SATA 8505 bacteriophage were isolated from Baltimore Inner Harbor waters using lysis of the Targeted Bacteria to form plaques in bacterial lawns as a means of detecting the presence of bacteriophage having lytic specificity for the Targeted Bacteria. Plaques were harvested, diluted, and re-plated on bacterial lawns through a process of serial enrichment until a single bacteriophage species, or monophase, was obtained as determined by a stable restriction fragment length profile of the bacteriophage DNA. The isolates obtained using the technique recited supra may be cultured using the techniques as set forth herein. The bacteriophage was deposited with the ATCC.

Example 2 Deposited Bacteriophage Concentration

Concentration of the Deposited bacteriophage may be determined using techniques known in the art (Adams, M. H. (1959). Methods of study bacterial viruses. Bacteriophages. London, Interscience Publishers, Ltd.; 443-519.). When a single phage particle encounters a permissive bacterium it will lyse it with the concomitant release of newly formed phage particles. When phages are mixed with host cells and poured in a layer of soft agar on the surface of a nutrient agar plate supporting bacterial growth, the cells will resume growth. In areas where no phages are present the bacteria will grow to stationary phase, forming a smooth opaque layer or lawn in the overlay. In areas where phages are present, phage progeny from each infected bacterium will infect neighboring bacteria, resulting in a growing zone of lysis full of liberated phage which eventually becomes visible to the naked eye as a plaque in the otherwise smooth bacterial lawn. These plaques can be counted, and their number is widely used for expressing phage titer in plaque-forming units or PFU. Using this approach, concentration of the Deposited bacteriophage may be determined. Briefly: (1) Various dilutions of the Deposited bacteriophage preparation are prepared; for example, by mixing 0.1 ml of phage sample with 9.9 ml of sterile LB broth. The samples are gently but thoroughly mixed. 0.5 ml of this mixture (which is a 10⁻² dilution of the original sample) is mixed with 4.5 ml of sterile LB broth (10⁻³ dilution). Several 10-fold dilutions are prepared in a similar fashion; (2) the contents of the tubes (1 ml of various dilutions) are transferred into sterile 10 ml culture tubes and 0.1 ml of host bacterial culture are added. The sample is mixed gently before proceeding immediately to the next step; (3) 3-5 ml of warm (45-50° C.) 0.7% LB agar (top agar) are added. The sample is mixed quickly and very gently. Then, the entire contents of the culture tube are poured onto a plate containing solidified LB agar (bottom agar). The plates are slid in circles a few times on the bench top immediately after pouring; (4) after sitting at room temperature for 10 min to allow the top agar to harden, the plates are inverted and placed into a 37° C. incubator and incubated overnight; (5) the next morning, the number of plaques on the plate with 30-300 individual well spaced plaques are counted and the titer calculated and expressed as PFU/ml of the starting sample.

Example 3 Production of the Deposited Bacteriophage

The Deposited bacteriophage is produced using a culture system. More specifically, strain of the host Targeted Bacteria or other closely-related bacterial species on which the bacteriophage can propagate is cultured in batch culture, followed by inoculation of the bacteriophage at the pre-determined multiplicity of infection (MOI). Following incubation and bacterial lysis, the bacteriophage is harvested and purified and/or concentrated to yield phage progeny suitable for the uses enumerated herein. Purification and concentration procedures included variously processing through filtration system(s), centrifugation (including continuous-flow centrifugation) or other well known bacteriophage purification and concentration techniques (Adams, M. H. (1959). Methods of study bacterial viruses. Bacteriophages. London, Interscience Publishers, Ltd.: 443-519.).

The invention provides compositions comprising active viral particles of the bacteriophage capable of lysing strains of Targeted Bacteria. The concentration of bacteriophage is determined using phage titration protocols. The final concentration of the bacteriophage is adjusted by concentration, if a more concentrated phage composition is desired, via filtration, centrifugation, or other means, or by dilution, if a less concentrated phage composition is desired, with water or buffer to yield a phage titer of 10⁶ to 10¹² PFU/ml, preferably 10¹⁰ to 10¹¹ PFU/ml. The resulting bacteriophage compositions are generally stored at 4° C.; alternatively, preparations can be freeze or spray-dried for storage, or can be encapsulated and stabilized with protein, lipid, polysaccharide, or mixtures thereof. Upon reconstitution, the phage titer can be verified using phage titration protocols and host bacteria. One of skill in the art is capable of determining bacteriophage titers using widely known bacteriophage assay techniques (Adams, M. H. (1959). Methods of study bacterial viruses. Bacteriophages. London, Interscience Publishers, Ltd.: 443-519.).

Example 4 Application of the Deposited Bacteriophage for Preservation of Food Products

The bacteriophage produced using the methods of the present invention may be dispersed in an appropriate aqueous solution or lyophilized or freeze-dried powder and applied to the surface of food products. Alternatively, the bacteriophage may be included with a cheese culture or other microbially active foodstuff prior to or during processing.

Example 5 Isolation of the Bacteriophage DNA

Bacteriophage DNA, a derivative of the bacteriophage, can be used for various applications such as for preparing DNA-based vaccines, and also for analytical purposes, for identifying the bacteriophage such as RFLP profile determination and comparison. Phage DNA can be isolated using a suitable commercial kit such as the Lambda Mini Kit (Qiagen, Inc.; Valencia, Calif.) or the standard phenol extraction technique. For example, 0.75 ml of phage in phosphate-buffered saline solution at a titer of 10⁸-10¹¹ PFU/ml is collected. 10 μl of Proteinase K (20 mg/ml) and 2 μl of RNAse (30 mg/ml) is added, followed by incubation at 37° C. for 30 minutes, and a subsequent incubation at 56° C. for 30 minutes. Following incubation, 75 μl of a mixture of 10% SDS (0.1 ml), 0.5 M EDTA (0.1 ml) and 0.8 ml of water is added and incubated at room temperature for 5 min. 0.75 ml of a phenol:chloroform:isoamylalcohol (25:24:1) solution is mixed well with the sample, followed by centrifugation at 13,000 RPM for five (5) min. Next, the supernatant (approximately 600 μl) is carefully removed and transferred to a clean eppendorf tube. 0.6 ml of chloroform is added to the supernatant, mixed well, and centrifuged at 13,000 RPM for five (5) min. The supernatant is then carefully extracted (approximately 500 μl). Next, 0.1 volumes of 3M sodium acetate (40 ml) is added to the solution, followed by 2.5 volumes of cold 95% ethanol (1 ml) to precipitate the bacteriophage DNA. The solution is allowed to incubate at −20° C. for 1 hour, followed by centrifugation at 13,000 RPM for thirty (30) min. Following centrifugation, the pellet is washed with 1 ml of 70% cold ethanol, and the supernatant is poured from the pellet. The pellet is allowed to air dry, and is then resuspended in 30-300 μl of TE (10 mM tris-HCL, pH=8.0-8.5, 1 mM EDTA).

Example 6 Restriction Fragment Length Polymorphism (RFLP) Profile

RFLP can be used to identify the Deposited bacteriophage or its progeny. The progeny will have a substantially equivalent RFLP DNA profile as the RFLP DNA profile of the original bacteriophage. A reference RFLP profile of the Deposited bacteriophage is shown in FIG. 1. DNA was isolated from, the bacteriophage using Qiagen Plasmid Miniprep or Midiprep kits (Valencia, Calif.) according to the manufacturer's directions. The DNA was quantitated by measuring absorbance at 260 nm. Approximately 0.5-1 μg of DNA was digested with an appropriate restriction enzyme (FIG. 1), and RFLP profile was determined on 1% agarose gel after staining with ethidium bromide.

Example 7 Lytic Specificity of the Bacteriophage

Sixty Staphylococcus aureus strains were screened for their susceptibility to the bacteriophage by the drop-on-lawn method, also known as the “spot test” method. The strains were streaked onto LB agar plates and incubated at 37° C. overnight. One colony of each strain was inoculated into a separate well of a 96-well microliter plate containing LB broth and incubated at 37° C. until the OD600 reached 0.2-0.3. One hundred microliters of each strain were mixed with LB soft agar and poured onto an LB agar plate. After the soft agar hardened 10 μl of the bacteriophage were spotted in triplicate onto the plates inoculated with the strains of Targeted Bacteria. Lytic activity was observed after overnight incubation at 37° C. The lytic specificity results are presented in Table 1. Of the sixty Staphylococcus aureus strains examined, 37, or 62%, were lysed by one or more of the deposited bacteriophage. The individual phage lysed from 10% to 38% of tested bacterial strains.

Example 8 Detection of Targeted Bacteria in Food Samples

The bacteriophage or its derivative, such as lytic enzyme, produced using the methods of the present invention is used to specifically lyse Targeted Bacteria without affecting any other prokaryotic or eukaryotic cells that may be present in the sample; thus, specifically eliciting their release of measurable bacterial products such as AK or ATP. Briefly: (1) Samples of the food to be analyzed are obtained and suspended in appropriate buffer, (2) The Deposited bacteriophage is added to the suspensions, as a result of which the Targeted Bacteria cells present in the samples are lysed and their ATP is released, (3) A luciferin + luciferase preparation is added to the mixtures, and (5) The mixtures' luminescence is measured within 60 sec, and the results are displayed on a handheld luminometer. It may be possible to establish a correlation between the luminometer readings and the number of Targeted Bacteria cells lysed (in general, the average amount of ATP per bacterial cell is 0.5-1.0 fg; precise correlation between the luminometer readings and the number of Targeted Bacteria cells should be experimentally established). If Targeted Bacteria cells are not present in the food samples analyzed, bacterial lysis and ATP-release will not occur.

Example 9 Preparing Vaccines and Bacterins

One example of utilizing bacteriophages to prepare vaccines and bacterins is to use the lytic Deposited bacteriophage to lyse specific strains of the Targeted Bacteria, which will yield bacterial lysates containing minimally-affected immunological epitopes of die bacteria. The phage may be removed from the final vaccine/bacterin preparation. Alternatively, it may be retained unaltered in the preparation because its lytic activity against Targeted Bacteria that may be present in the mammalian organism being vaccinated may increase the preparation's efficacy. In one preferred embodiment of the present invention: (i) the most prevalent, problematic strains of the Targeted Bacteria are chosen so that the vaccine/bacterin contains the immunological epitopes that are most, relevant for protecting against the infection, and (ii) the bacteriophage is kept unaltered in the final vaccine/bacterin, at levels ranging from 10⁶-10 ¹⁰ PFU/ml.

Bacteriophage-based vaccines and bacterins also may be prepared by using derivatives of the Deposited bacteriophage to lyse the Targeted Bacteria. An example of the general methodology can be briefly outlined from a recent study (Panthel, K., W. Jechlinger, et al. (2003). “Helicobacter pylori ghosts by PhiX protein E-mediated inactivation and their evaluation as vaccine candidates.” Infect Immun 71(1): 109-16.) of an Helicobacter pylori bacterin. The authors used E. coli-H. pylori shuttle plasmid pHe12 and lysis gene e of bacteriophage φX174 to construct H. pylori lysis plasmid pHPC38, which they introduced into H. pylori strain P79. At a pre-determined time, the authors triggered e gene-expression in order to elicit bacterial lysis, and they found that the lysate protected BALB/c mice against H. pylori infection.

The above description of various illustrated embodiments of the invention is not intended to be exhaustive or to limit the invention to the precise form disclosed. While specific embodiments of, and examples for, the invention are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the invention, as those skilled in the relevant art will recognize. The invention may be practiced in ways other than those particularly described in the foregoing description and examples. The teachings provided herein of the invention can be applied to other purposes, other than the examples described below.

The entire disclosure of each document cited (including patents, patent, applications, journal articles, abstracts, manuals, books, or other disclosures) in the Background of the Invention, Detailed Description, and Examples is herein incorporated by reference in their entireties. 

1. An isolated bacteriophage equivalent to the bacteriophage SAML-4, SAML-150, or SATA-8505 deposited under ATCC accession Nos. PTA-9474, PTA-9475, and PTA-9476, respectively, wherein said bacteriophage has lytic activity against Staphylococcus aureus strains.
 2. Isolated progeny of the bacteriophage of claim 1, wherein said progeny of said bacteriophage have the same phenotypic characteristics and same lytic activity against Staphylococcus aureus strains as said bacteriophage.
 3. A composition comprising an isolated bacteriophage of claim
 1. 4. A composition comprising an isolated bacteriophage of claim
 2. 5. At least one isolated derivative of the bacteriophage of claim 1, wherein said derivative comprises nucleic acids, partial or complete genes, gene expression products, structural components, or one or more combinations thereof.
 6. At least one isolated derivative of the bacteriophage of claim 2, wherein said derivative comprises nucleic acids, partial or complete genes, gene expression products, structural components, or one or more combinations thereof.
 7. The isolated bacteriophage of claim 1, wherein said Staphylococcus aureus strain is a MRSA (MDRSA) strain.
 8. The isolated progeny of claim 2, wherein said Staphylococcus aureus strain is a MRSA (MDRSA) strain. 